Chemiluminescence proximity nucleic acid assay

ABSTRACT

This invention relates to the detection and quantitation of target nucleic acids in a heterogeneous mixture in a Sample and the methods of use thereof. The detection system includes a chemiluminescent molecule, a chemiluminescent substrate, a dye that is light responsive when intercalated into nucleic acids and nucleic acids. This invention is useful in any application where detection of a specific nucleic acid sequence is desirable, or where the detection of enzymes that modify nucleic acids is desirable such as diagnostics, research uses and industrial applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject application claims the benefit of priority to U.S.Application Ser. No. 60/687,647, filed on Jun. 3, 2005, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to the detection and quantitation of targetnucleic acids in a heterogeneous mixture in a Sample and the methods ofuse thereof. The detection system includes a chemiluminescent molecule,a chemiluminescent substrate, a dye that is light responsive whenintercalated into nucleic acid and a nucleic acid target. The methodrequires that a specific three-dimensional structure (i.e. Dyeintercalated into nucleic acid) be created for energy to be accepted bythe dye and that the energy donor (Chemiluminescent Molecule) beproximal to this structure. This invention is useful in any applicationwhere detection of a specific nucleic acid sequence is desirable, orwhere the detection of enzymes that modify nucleic acids is desirablesuch as diagnostics, research uses and industrial applications.

BACKGROUND OF THE INVENTION

Nucleic acids are measured to identify molecules of a specific targetnucleic acid sequence in a population of heterogeneous nucleic acids,DNA or RNA, or to measure products of reactions where nucleic acids, DNAor RNA, are modified. Such measurements are generally permutations ofthe following procedures:

a. where the starting nucleic acid is RNA, conversion to DNA isaccomplished by a reverse transcription reaction. The oligonucleotideprimers for the reverse transcription reaction may be specific for thetarget sequence or may be general for conversion of all RNA sequences toDNA;

b. amplification of the target nucleic acid by target sequence specificreactions. These include polymerase chain reaction (PCR) with sequencespecific primers, and primer extension reactions again with a targetsequence specific oligonucleotide primer. Rolling circle amplificationof DNA has also been used to amplify specific DNA sequences;

c. physical separation of the heterogeneous nucleic acids. Such physicalseparations include but are not limited to size fractionation andaffinity separation when amplified nucleic acids are produced withderivatized substrates including but not limited to biotinylateddeoxyribonucleotide triphosphates;

d. labeling of the nucleic acid. As mentioned in c. above, amplifiednucleic acids may be labeled using either derivatizeddeoxyribonucleotide triphosphates or derivatized oligonucleotide (RNA orDNA) primers; and

e. detection of the nucleic acids. Nucleic acids can be detected eitherthrough the labeling moiety, or by physical separation followed bydetection with nucleic acid specific dyes.

One of the more common methods for the quantitative detection of targetsequences is the sequence specific amplification of the targetsequence(s) by PCR, either from DNA or from cDNA after reversetranscription, physical separation by gel or capillary electrophoresis,and detection by fluorescent labeling (e.g. of dsDNA by ethidium bromideor by use of fluorescently labeled primers in the amplification).Another common technique for the quantitative detection of targetsequence(s) involves “real time” PCR.

PCR technology is widely used to aid in quantitating DNA because theamplification of the target sequence allows for greater sensitivity ofdetection than could otherwise be achieved. The point at which thefluorescent signal is measured in order to calculate the initialtemplate quantity can either be at the end of the reaction (endpointQPCR) or while the amplification is still progressing (real-time QPCR).The more sensitive and reproducible method of real-time QPCR measuresthe fluorescence at each cycle as the amplification progresses.

The reporter molecule used in real-time QPCR reactions can be (1) asequence-specific probe composed of an oligonucleotide labeled with afluorescent dye plus a quencher or (2) a non specific DNA binding dyethat fluoresces when bound to double stranded DNA.

Both of these techniques, and others not described in detail, requireinstrumentation either for physical separation or detection. Therequirement for instrumentation and/or separation technologies withtheir attendant sample handling limits the use of quantitative andqualitative target sequence detection. Accordingly, there is a need formethods of detecting and measuring nucleic acids that do not requireexpensive, delicate instrumentation either for sample separation or fordetection. Such measurements include but are not limited to theidentification of molecules of a specific nucleic acid sequence as wellas the detection of nucleic acids that are the product of nucleic acidmodifying reactions. Nucleic acid modifying reactions include but arenot limited to polymerization reactions, ligation reactions, nucleasereactions and recombination reactions.

Fluorescent Intercalating Nucleic Acid Dyes

A common method for the detection of nucleic acids is by staining themwith fluorescing intercalating dyes. These dyes have several uniquefeatures that make them especially useful: 1) They have a high molarabsorptivity; 2) Very low intrinsic fluorescence: 3) Large fluorescentenhancements upon binding to nucleic acids; and 4) Moderate to highaffinity for nucleic acids, with little or no staining to otherbiopolymers. Intercalating nucleic acid stains have fluorescenceexcitations and emissions that span the visible-light spectrum from blueto near-infrared with additional absorption peaks in the UV, making themcompatible with many different types of instrumentation. These dyes areexcited with an extrinsic light source that has a spectrum that overlapswith the maximally excitation wavelength of the intercalated dye. Theymay be used to image both RNA and DNA. Some commonly used dyes arelisted below.

Dye Name Ex/Em* Application Ethidium Bromide 300/600 Quantitation andDetection of dsDNA Ethidium Bromide 510/620 Quantitation and Detectionof dsDNA Homodimer-1 PICOGREEN ® 502/523 dsDNA Quantitation ReagentOLIGREEN ® 498/518 Quantitation and Detection of ssDNA and QuantitationReagent oligonucleotides RIBOGREEN ® 500/520 Quantitation and Detectionof RNA Quantitation Reagent SYBR GOLD ® stain 495/537 Quantitation andDetection of single- or double-stranded DNA or RNA post- electrophoresisSYBR GREEN I ® stain 494/521 Quantitation and Detection ofdouble-stranded DNA and oligonucleotides post- electrophoresis Alsouseful for real-time PCR assays SYBR GREEN ® stain 492/513 Sensitivestain for RNA and single-stranded DNA post-electrophoresis SYBR SAFE ®stain 502/530 Sensitive DNA gel stain with significantly reducedmutagenicity SYBR DX DNA BLOT ® 475/499 Sensitive stain for DNA stain*Excitation (Ex) and emission (Em) maxima are the wavelength, innanometers, (nm) of light that maximally excites the intercalated dyeand the wavelength of light that is maximally emitted when the dyefluoresces, respectively.

Resonance Energy Transfer

Energy may be donated to nucleic acid intercalated dye either by photonsor by resonance energy transfer. The principle of energy transferbetween two molecules can be exploited as a means to provide informationabout relative changes in their proximity and orientation to oneanother. Resonance Energy Transfer (RET) is the transfer of excitedstate energy from a donor to an acceptor molecule. Förster resonanceenergy transfer (FRET) is a distance-dependent interaction between theelectronic excited states of two dye molecules in which excitation istransferred from a donor molecule to an acceptor molecule withoutemission of a photon. This can only occur if the absorption spectrum ofacceptor molecule overlaps with the emission spectrum of the donor.Förster determined that the degree of resonance energy transfer betweenthe energy donor and energy acceptor is inversely proportional to thedistance between the two molecules to the sixth power. In the case ofFRET, an external light source of specific wavelength is used to excitethe donor molecule.

Bioluminescent Resonance Energy Transfer (BRET) uses biologicalmolecules such as a luciferase as the donor molecule. Depending on thespecies of origin, luciferases that use coelenterazine as a substrategenerate blue light in the range of 450 to 500 nm. When a suitableacceptor is in close proximity, the blue light energy is captured byRET. The acceptor molecules are generally a class of proteins that haveevolved the ability to be excited by blue light and then fluoresce inlonger wavelengths typically with maximal spectral emissions above 500nm. In both FRET and BRET the molecules of interest may be eithercovalently or non-covalently linked or brought in to proximity byconformational change or by spatial migration or by an alteration intheir relative orientations to one another. For instance, the twomolecules may be conjugated to two separate proteins of interest. Theymay then be brought into proximity by their affinity for one another ortheir affinity for a third molecule. They may also be attached to aprotein of interest and then brought closer due to a conformationalchange within the protein of interest. Generally the two molecules mustbe within 100 Å of one another for resonance energy transfer to occurand changes as little as 1-2 Å may be detected. Luciferases that havebeen used in BRET include those from the firefly, Renilla reniformis andGaussia princeps. A commonly used fluorescent protein is the greenfluorescent protein (GFP) from Aequorea victoria. BRET is generally usedto measure the degree of affinity or degree of conformational changebetween two protein domains either covalently or non-covalently linked.

SUMMARY OF THE INVENTION

The present invention functions to bring a Chemiluminescent Moleculewithin close proximity to dye stained target nucleic acids or theproducts of nucleic acid modifying reactions. The ability of the energyto be accepted by the dye is conditional. It is necessary for the dye tobe intercalated into nucleic acid and that the energy donor be in closeproximity. The method requires that a specific three-dimensionalstructure (i.e. Dye intercalated into nucleic acid that is contacted toa Chemiluminescent Molecule) be created for energy to be accepted by thedye and that the energy donor be proximal to this structure.

Specific advantages of the present invention include the following. Theinvention is rapid, and does not require any wash steps, which issignificant as would be recognized by one of skill in the art. It doesnot require radioactivity nor does it require a laser for activatingnucleic acid conjugated fluorophores. The signal from the emitted lightin the reactions may be integrated over minutes as opposed tomilliseconds as is the case with laser activated fluorophores.

A unique aspect of this method is that of Proximity. Direct contact ofthe Chemiluminescent Molecule to the nucleic acid allows for thesensitive detection of a change in the mass of stainable nucleic acid(Example 3). The amount of fluorescence from nucleic acid that has beenstained with an intercalating dye is directly proportional to the amountof nucleic acid present. Any condition in which the total mass ofnucleic acid that is attached to the Chemiluminescent Molecule isincreased or decreased will result in an increase or decrease offluorescence by an activated intercalating dye.

The Chemiluminescent Molecule does not simply act as an indicator of thepresence of contact of a probe to a fluorophore. It indicates thatduplex nucleic acid is present by virtue of its illumination of dyebound that can only act as an energy acceptor when bound to duplex. Inthe case of detecting nucleic acids of specific sequence it adds a levelof stringency. A positive signal requires both that the indicatormolecule (i.e. the Chemiluminescent Molecule) be associated with thetarget sequence and also that nucleic acid be present. In other words itdemands that a specific three-dimensional structure be created forenergy to be accepted by the dye and that the energy donor be a part andthus proximal to this structure. This will significantly reduce thebackground noise in the system for which it is being applied.

The presence of the target nucleic acid is conveyed when the lightemitting Chemiluminescent Molecule is brought into close proximity inthe presence of fluorescent intercalated dye. The light emitted by theintercalated dye is proportional to the amount of stainable nucleic acidthat is in close proximity to the Chemiluminescent Molecule.

The present invention relates to a general detection system for nucleicacids and methods of use thereof. The preferred system comprises fourreagents: 1) a Chemiluminescent Molecule, 2) a ChemiluminescentSubstrate, 3) an Intercalating Dye and 4) Nucleic Acid. These reagentsare contacted with a Sample and can detect a change in the mass ofstainable nucleic acid caused hybridization to complementary nucleicacids or by nucleic acid modifying reactions. The nucleic acids in aSample can be either unamplified or the result of amplificationreactions.

A Chemiluminescent Probe may be made by covalently or non-covalentlyattaching the Chemiluminescent Molecule to a single stranded nucleicacid probe capable of hybridizing to complementary single strandednucleic acid in the Sample. The target nucleic acid being probed in theSample may be in solution phase with Chemiluminescent Probe in solutionphase being added. The nucleic acid being probed in the Sample may beimmobilized on a solid support with the Chemiluminescent Probe insolution phase being added. The Chemiluminescent Probe may beimmobilized on a solid support with the nucleic acid being probed in theSample in solution phase being added. The Chemiluminescent Probe and thenucleic acid being probed in the Sample may be immobilized.

The Intercalating Dye is added to the Sample containing double strandednucleic acid and a Chemiluminescent Molecule or Probe and itintercalates into the double stranded nucleic acid regions in theSample. The Chemiluminescent Substrate is added to the Sample and isactivated by the Chemiluminescent Molecule. The interaction of theChemiluminescent Molecule and Chemiluminescent Substrate produces energythat in turn excites the Intercalating Dye at the Intercalating DyeExcitation Wavelength and the Intercalating Dye emits light at theIntercalating Dye Emission Wavelength. The light emitted at theIntercalating Dye Emission Wavelength is measured (with or withoutappropriate emission filters) and it is possible to determine thepresence and quantitate the amount of target nucleic acid in the Sample.Using a filter one may discriminate longer wavelength light emitted bythe fluorescing intercalated dye from the shorter wavelength lightemitted by the Chemiluminescent Molecule. This discrimination may alsobe accomplished by incorporating into the Chemiluminescent Reactionnon-intercalating, non-fluorescing dyes that absorb light emitted at thewavelengths produced by the Chemiluminescent Molecule but not that ofthe fluorescent intercalated dye. This general method is depicted inFIG. 1.

In one non-limiting embodiment, the Sample contains single strandedgenomic DNA suspected of containing integrated HIV proviral sequence.The Chemiluminescent Molecule is Gaussia princeps luciferase (gluc) andit is covalently attached to a ssDNA probe that is complementary to aregion of the HIV envelope gene. The Intercalating Dye is PICOGREEN® andthe Chemiluminescent Substrate is coelenterazine. The Sample DNA isdenatured to generate single strands and then the probe covalentlyattached to Gaussia luciferase is added to the Sample and hybridizes toits complement. The PICOGREEN® is added to Sample and intercalates intothe dsDNA region resulting from the probe hybridization. Thecoelenterazine is added to the Sample and causes the Gaussia luciferaseto emit blue light with a spectrum peak at 480 nm. The emitted bluelight causes any intercalated PICOGREEN® in close proximity to beexcited, since its peak excitation wavelength is 502 nm. The PICOGREEN®then emits a bright green spectrum of light with a peak at 523 nm thatcan be easily measured with a charged coupling device (CCD) camera thatis equipped with a filter that significantly diminishes wavelengthsbelow 500 nm.

An additional nonlimiting disclosure of the present invention wouldcreate a proximity assay by bringing a chemiluminescent molecule intoclose proximity with nucleic acid polymers incorporating fluorescentlylabeled nucleotides, or nucleotide analogs that fluoresce, in place ofthe intercalating dyes of the present invention. U.S. Pat. Nos.6,451,536 and 6,960,436 describe the use of fluorescent nucleotides todetect and measure DNA samples without the component of proximity thatembodies the present invention. These above referenced patents arehereby incorporated in their entirety by reference.

This invention is useful in any application where detection of thepresence or absence of DNA is desirable, such as diagnostics, researchuses and industrial applications. This method is particularly wellsuited to detecting DNA in Samples either in solution or in a microarrayformat. This method is also well suited to detecting the products ofenzymatic activities that create or modify nucleic acid samples such aspolymerases, nucleases, recombinases and ligases as well detectinginhibitors of these activities. The present invention also encompassesmethods of use of the above-described system.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an embodiment having Probe directly attached toChemiluminescent Molecule and Sample where both are unattached to aSolid Support.

FIG. 2 depicts an embodiment having Probe directly attached to aChemiluminescent Molecule and Sample is immobilized on Solid Support.

FIG. 3 depicts an embodiment having Probe indirectly attached to aChemiluminescent Molecule and Sample both are unattached to a SolidSupport.

FIG. 4 depicts an embodiment having Probe indirectly attached to aChemiluminescent Molecule and Sample is immobilized on Solid Support.

FIG. 5A shows the CCD camera images for reactions described in Example 1when SYBR GREEN I® is the Intercalating Dye. In this experiment theluciferase and SYBR GREEN I® concentrations are held constant while theDNA concentration is varied.

FIG. 5B shows the data generated in Example 1 presented as relativeintensity per spot.

FIG. 6A shows the CCD camera images for reactions described in Example 2when SYBR GREEN I® is the Intercalating Dye. In this experiment theluciferase and DNA concentrations are held constant while the SYBR GREENI® concentration is varied.

FIG. 6B shows the data generated in Example 1 presented as relativeintensity per spot when SYBR GREEN I® is the Intercalating Dye.

FIG. 7A shows a schematic diagram of the experiment described in Example3 where the biotinylated gluc is brought into close proximity to thebiotinylated DNA duplex by a streptavidin intermediate in the presenceof SYBR GREEN I®.

FIG. 7B shows the data generated in Example 3 where the biotinylatedgluc is brought into close proximity to the biotinylated DNA duplex by astreptavidin intermediate in the presence of SYBR Green I®.

FIG. 7B shows the data generated in Example 3 presented as relativeintensity per spot when SYBR GREEN I® is the Intercalating Dye.

FIG. 8 shows the predicted data from a hypothetical assay using theGaussia princeps luciferase conjugated to a DNA oligomer probe toquantitate DNA of a unique sequence in mixed sample.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a general method for detecting the presence orabsence of nucleic acid in a Sample. In a preferred embodiment, thesystem comprises four reagents: 1) a Chemiluminescent Molecule, 2) aChemiluminescent Substrate, 3) an Intercalating Dye and 4) NucleicAcids. The following terms are intended to have the following generalmeanings as they are used herein as would be readily understood by oneof skill in the art.

A. DEFINITIONS

“Bioluminescent Molecule” means any biological molecule involved in achemiluminescent reaction. The reaction may be either catalytic orstoichiometric.

“Chemiluminescent Emission Spectrum” means the range of photonwavelengths emitted by the Chemiluminescent Molecule. The spectrum isfrequently defined by the wavelength of highest intensity from achemiluminescent reaction.

“Chemiluminescent Probe” means an olio- or poly-nucleotide probemolecule with a coupled Chemiluminescent Molecule. The ChemiluminescentMolecule may be coupled covalently or through non-covalent interaction,either before or after modification of the Probe by target nucleic acid.

“Chemiluminescent Substrate” means a reactant that interacts with theChemiluminescent Molecule to produce a photon/light.

“Chemiluminescent Molecule” means any molecule that takes part in anychemiluminescent reaction; this includes but is not limited to abioluminescent molecule.

Various Chemiluminescent Molecules and their respective ChemiluminescentSubstrates include but are not limited to:

-   -   i) Luciferases that utilize coelenterazine as a Substrate        including luciferases from the organisms Gaussia princeps,        Periphylla periphylla, Renilla mulleri and Aequorea Victori.    -   ii) Firefly luciferase that utilizes firefly luciferin as        Substrate.    -   iii) Alkaline phosphatase that utilizes DuoLuX™        Chemiluminescent/Fluorescent Substrate for phosphatase.    -   iv) Horseradish peroxidase that utilizes DuoLuX™        Chemiluminescent/Fluorescent Substrate for peroxidase

“Chemiluminescent Reaction” means any chemical reaction that produces aphoton without an input photon. The reactants may act eithercatalytically or stoichiometrically. In the case of a catalyticreaction, the catalyst converts a substrate(s) into a product(s) withthe concomitant release of a photon. In the case of a stoichiometricreaction, two or more reactants are converted to product(s) and aphoton.

“Complementary base pairs” means the purine and pyrimidine bases thatpair to form stable hydrogen bonds between two single strand nucleicacid molecules. The usual base pairs are adenine and thymidine, guanineand cytosine, and adenine and uracil. Other base pairs includederivatized variants of these bases, including but not limited tomethylated bases, and other purines and pyrimidines including but notlimited to inosine.

“Double strand nucleic acid” means two single strand nucleic acidmolecules that are non-covalently associated by hydrogen bonding ofcomplementary bases on the two molecules.

“Excitation” means the transfer of energy from a ChemiluminescentMolecule to the Intercalating Dye. Energy transfer from a luminescentmolecule to the Intercalating Dye may be through the donation of photonsor through Resonance Energy Transfer (RET).

“Hybridization” means the association reaction between two nucleic acidmolecules through complementary base pairs to form a double strandnucleic acid.

“Intercalating Dye” means a molecule that binds to double stranded orsingle stranded nucleic acids between adjacent base pairs. Further, uponintercalation the dye undergoes a change in its electronic configurationsuch that its absorption and/or emission spectra change. The dye has avery low intrinsic fluorescence when not bound to nucleic acids. The dyehas a very large enhancement of fluorescence upon binding to nucleicacids with increases in quantum yields to as high as 0.9. The dye has avery high affinity for nucleic acids and little or no staining of otherbiopolymers.

“Intercalating Dye Excitation Spectrum” means the range of wavelengthsof energy that excites an intercalated dye complexed with doublestranded or single stranded nucleic acid to produce a photon at itsemission spectrum. The Intercalating Dye Excitation Spectrum overlapswith the emission spectrum of the Chemiluminescent Molecule.

“Intercalating Dye Emission Spectrum” means the wavelengths of photonsemitted by intercalated dye complexed with double stranded or singlestranded nucleic acid when excited by a light source with a spectrumthat overlaps with its maximal excitation wavelength.

“Nucleic acid” means an oligomer or polymer of DNA, RNA or a chimera ofboth. It includes oligomers or polymers of DNA, RNA or chimeras of bothinto which analogs of nucleotides have been incorporated. It alsoincludes oligomers and polymers of nucleotide analogs, as would berecognized by one of skill in the art. Examples of nucleotide analogsinclude nucleotides such as Locked Nucleic Acid (LNA) or Peptide NucleicAcid (PNA) or other nucleotide analogs that are capable of complementarybase-pairing with DNA or RNA, or nucleotide analogs that can beincorporated by enzymes that modify DNA such as telomerases, DNApolymerases, DNA repair enzymes, reverse transcriptases, or DNA and RNAligases, or other DNA modifying enzymes known to those skilled in theart.

“Probe” means any single strand nucleic acid with a defined sequence ofpurine and pyrimidine bases, including modifications as would berecognized by one of skill in the art.

“Proximity” means the condition in which different molecules are closeby virtue of their association in a stable molecular complex as would beappreciated by one of skill in the art. The molecules may be associatedthrough covalent or non-covalent interactions. It is envisioned that thesize of such complexes would be at the level seen in mostprotein/protein, protein/nucleic acid and nucleic acid/nucleic acidcomplexes. The proximity of the Chemiluminescent Molecule to nucleicacid would preferably be less than 500 Å. The proximity of theChemiluminescent Molecule to nucleic acid would more preferably be lessthan 250 Å. The proximity of the Chemiluminescent Molecule to nucleicacid would most preferably be less than 100 Å. The nucleic acid may havea length much greater than 500 Å.

“Sample” means any mixture of molecules collected from solid, solutionor gas that may contain nucleic acid or activity that may modify nucleicacid or inhibitors of said activity.

“Single strand nucleic acid” means an oligomer or polymer of repeatingunits of phosphate and ribose or deoxyribose joined at the 3′ and 5′positions of the sugar rings together with the purine or pyrimidinebases attached at the position of the ribose or deoxyribose ring.

“Solid support” includes any suitable support for a binding reactionand/or any surface to which molecules may be attached through eithercovalent or non-covalent bonds. This includes, but is not limited to,membranes, plastics, paramagnetic beads, charged paper, nylon,Langmuir-Blodgett films, functionalized glass, germanium, silicon, PTFE,polystyrene, gallium arsenide, gold and silver. Any other material knownin the art that is capable of having functional groups such as amino,carboxyl, thiol or hydroxyl incorporated on its surface, is alsocontemplated. This includes surfaces with any topology, including, butnot limited to, flat surfaces, spherical surfaces, grooved surfaces, andcylindrical surfaces e.g., columns. Probes may be attached to specificlocations on the surface of a solid support in an addressable format toform an array, also referred to as a “microarray” or as a “biochip.”

B. THE GENERAL METHOD (THE ILLUSTRATIVE EMBODIMENTS ARE NOT EXHAUSTIVEOF THE EMBODIMENTS DISCLOSED IN THE PRESENT INVENTION)

The preferred embodiment of the present invention comprises fourmolecules: the first is a Chemiluminescent Molecule, the second is aChemiluminescent Substrate, the third is an Intercalating Dye and thefourth is Nucleic Acids. The absorption spectrum of the IntercalatingDye overlaps with the emission spectrum of the ChemiluminescentMolecule.

In one embodiment, the Chemiluminescent Molecule is linked, covalentlyor noncovalently, to a single strand nucleic acid complementary to thetarget sequence; this will be called the “Probe”. When the “Sample”nucleic acid is denatured and allowed to reanneal in the presence of the“Probe”, the “Probe” and the target sequences in the Sample will formdouble stranded DNA. This double stranded DNA will in turn associatewith the Intercalating Dye. The intercalation of the Intercalating Dyeinto double stranded will shift the absorption spectrum of theIntercalating Dye to overlap with the emission spectrum of theChemiluminescent Molecule.

Finally, when the Chemiluminescent Molecule is provided withChemiluminescent Substrate, it will generate the energy to excite theIntercalated Dye molecules and in turn cause them to emit photons attheir emission wavelengths. These photons can be detected/counted. Onemethod to quantitate the light emitted by the dye is to apply a filterthat is able to discriminate between light emitted at the lowerwavelength from light emitted by the intercalated dye. The efficiencywith which energy produced by the Chemiluminescent Molecule is capturedby the intercalated Intercalating Dye molecules will depend on thedistance between them. The light emitted by the Intercalating Dye is afunction the distance between the light source (ChemiluminescentMolecule) and the Intercalating Dye. If Excitation by theChemiluminescent Molecule occurs by Resonance Energy Transfer thenForster's Equation applies. Forster's Equation states that the transferof excitation energy between the donor (Chemiluminescent Molecule suchas luciferase) and acceptor (Intercalating Dye such as PICOGREEN®) dropsoff as the 6^(th) power of the distance between the two.

An advantage of the present invention is that no light source aside fromthe Chemiluminescent Molecule is necessary for detection. Further, theassociation of the Chemiluminescent Probe with double strand DNA can bemeasured without physical separation of the target from other doublestrand nucleic acid, as only double strand DNA with intercalatedIntercalating Dye by close physical association with theChemiluminescent Probe will produce signal over background. This aspectof the invention alleviates the need for washes, a significant advantageas would be recognized by one with skill in the art. Any detector thatcan discriminate between the shorter and longer spectra wavelengths canbe utilized in this assay system. These include, but are not limited toluminometers, fluorimeters, and CCD cameras equipped with a filter toremove shorter wavelengths in the range of that emitted by theChemiluminescent Molecule.

C. EMBODIMENT HAVING PROBE DIRECTLY LABELED WITH CHEMILUMINESCENTMOLECULE AND SAMPLE IN SOLUTION

FIG. 1 is a schematic representation of a Chemiluminescent Probe beingused to quantitate nucleic acid of specific sequence in solution phase.Here the Chemiluminescent Molecule is a luciferase. The luciferase iscovalently attached to a ssDNA probe. This Bioluminescent Probe is addedto a sample containing target sequence nucleic acid in solution and anucleic acid stain. Coelenterazine is then added to activate theluciferase. The luciferase excites the nucleic acid intercalated stainthat in turn emits a spectrum of light with a maximal wavelength of 520nm. Light with wavelengths below 500 nm is filtered out. Light withwavelengths greater than 500 nm is permitted to pass to a detector.

D. EMBODIMENT HAVING PROBE DIRECTLY LABELED WITH CHEMILUMINESCENTMOLECULE AND SAMPLE IS IMMOBILIZED ON SOLID SUPPORT

FIG. 2 is a schematic representation of a Chemiluminescent Probe beingused to quantitate nucleic acid of specific sequence immobilized on asolid support. Here the Chemiluminescent Molecule is a luciferase. Theluciferase is covalently attached to a ssDNA probe. This BioluminescentProbe is then added to the Sample with target nucleic acid immobilizedin a solid support. Nucleic acid stain is present in solution in theSample. Coelenterazine is then added to activate the luciferase. Theluciferase excites the nucleic acid intercalated stain that in turnemits a spectrum of light with a maximal wavelength of 520 nm. Lightwith wavelengths below 500 nm is filtered out. Light with wavelengthsgreater than 500 nm is permitted to pass to a detector.

E. EMBODIMENT HAVING PROBE INDIRECTLY LABELED WITH CHEMILUMINESCENTMOLECULE AND SAMPLE IN SOLUTION

FIG. 3 is a schematic representation of a Chemiluminescent Probe beingused to quantitate nucleic of specific sequence in solution phase. Herethe Chemiluminescent Molecule is a luciferase. The luciferase isnoncovalently conjugated to a biotinylated ssDNA probe through astreptavidin intermediate. Because a single streptavidin molecule maybind four biotin molecules, biotinylated probe DNA, biotinylatedluciferase and streptavidin may be mixed in the appropriate ratios togenerate a Bioluminescent Probe. The Bioluminescent Probe is added to asample containing target sequence nucleic acid and a nucleic acid stain.Coelenterazine is added to activate the luciferase. The luciferase thenexcites the nucleic acid intercalated stain, which in turn emits aspectrum of light with a maximal wavelength of 520 nm. Light withwavelengths below 500 nm is filtered out. Light with wavelengths greaterthan 500 nm is permitted to pass to a detector.

F. EMBODIMENT HAVING PROBE INDIRECTLY LABELED WITH CHEMILUMINESCENTMOLECULE AND SAMPLE IS IMMOBILIZED ON SOLID SUPPORT

FIG. 4 is a schematic representation of a Chemiluminescent Probe beingused to quantitate nucleic acid of specific sequence immobilized on asolid support. Here the Chemiluminescent Molecule is a luciferase. Theluciferase is noncovalently conjugated to a biotinylated ssDNA probethrough a streptavidin intermediate. Because a single streptavidinmolecule may bind four biotin molecules biotinylated probe DNA,biotinylated luciferase and streptavidin may be mixed in the appropriateratios to generate a Bioluminescent Probe. This Bioluminescent Probe isthen added to the Sample with target nucleic acid immobilized in a solidsupport. Nucleic acid stain is present in solution in the Sample.Coelenterazine is then added to activate the luciferase. The luciferaseexcites the nucleic acid intercalated stain, which in turn emits aspectrum of light with a maximal wavelength of 520 nm. Light withwavelengths below 500 nm is filtered out. Light with wavelengths greaterthan 500 nm is permitted to pass to a detector.

G. EMBODIMENT HAVING PROBE DIRECTLY LABELED WITH CHEMILUMINESCENTMOLECULE, PROBE IMMOBILIZED ON SOLID SUPPORT AND SAMPLE IN SOLUTION H.USES OF THE INVENTION

The invention is a general method for detecting and quantitating targetnucleic acid sequences in a heterogeneous mixture of nucleic acids. Thedetection of nucleic acids is important for many applications, including(but not limited to) diagnostic measurements of nucleic acids in bodilytissues and fluids as would be readily understood by one of skill in theart.

The method serves to monitor the increase or decrease of stainablenuclei acid that is contacted to a Chemiluminescent Molecule. Stainablenucleic acid is any polymer of nucleic acid into which IntercalatingDyes will incorporate as opposed to other biological molecules. Uponbinding, these Intercalating Dyes undergo a change in their electronicconfiguration that makes them fluoresce in the presence of theappropriate excitation wavelength.

This will occur when the mass of stainable nucleic acid that iscontacted to the Chemiluminescent molecule is altered. This includes butis not limited to the following:

-   -   a. The method measures enzymatic activity that polymerizes the        extension of a nascent strand, through the incorporation of        nucleotides or nucleotide analogs, of nucleic acid when the        extended strand or in the case of duplex its complement are        contacted to a Chemiluminescent Molecule and a light responsive        intercalating dye is present. Said activity includes but is not        limited to RNA polymerases, DNA polymerases and telomerases. The        invention also serves to detect inhibitors of the activities        thereof;    -   b. The method measures enzymatic activity that degrades nucleic        acid when the nucleic acid is contacted to a Chemiluminescent        Molecule and a light responsive intercalating dye is present.        Said activity includes but is not limited to RNA exonucleases,        RNA endonucleases, DNA exonucleases and DNA endonucleases. The        invention also serves to detect inhibitors of the activities        thereof;    -   c. The method measures enzymatic activity that facilitates the        attachment or ligation of separate nucleic acid molecules when        one of the nucleic acid molecules is contacted to a        Chemiluminescent Molecule and a light responsive intercalating        dye is present. Said activity includes but is not limited to DNA        ligases. The invention also serves to detect inhibitors of the        activities thereof;    -   d. The method measures enzymatic activity that facilitates the        recombination of nucleic acid duplex molecules when one of the        nucleic acid duplex molecules is contacted to the        Chemiluminescent Molecule, a light responsive intercalating dye        is present and the mass of the nucleic acid duplex of the        recombined product is different than the mass of the nucleic        acid duplex of the non-recombined molecule. Said activity        includes but is not limited to recombinases and integrases. The        invention also serves to detect inhibitors of the activities        thereof;    -   e. The method measures enzymatic activity that facilitates the        attachment or ligation of duplex nucleic acid molecules to        protein molecules when the protein molecules are contacted to or        are a Chemiluminescent Molecule and a light responsive        intercalating dye is present. The invention also serves to        detect inhibitors of the activities thereof.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety. The following examplesserve to illustrate certain preferred embodiments and aspects of thepresent invention and are not to be construed as limiting the scopethereof.

EXAMPLES Example One

Luciferase activation of DNA intercalated dye is proportional to the DNApresent.

Objective:

The objective of this experiment was to determine if the luciferaseenzyme of Gaussia princeps (gluc) is sufficient to act as an excitationsource for a fluorophore that is staining double stranded (ds) DNA.Specifically, the experiment is intended to determine if the gluc whichemits light at a peak of 480 nm can excite a dsDNA intercalated nucleicacid stain with an excitation maximum in the range of 495 nm to 500 nmand an emission maximum of approximately 520 nm. This would be done bydetecting light from a gluc, fluorescing nucleic acid stain/dsDNAmixture which has had wavelengths below 500 nm filtered out.

Materials and Methods:

The Gaussia princeps luciferase was from Avidity LLC (Denver, Colo.).The SYBR Green I nucleic acid stain and the linearized dsDNA ladder wereobtained from Invitrogen (Carlsbad, Calif.).

The reactions for the detection of dsDNA were as follows. Dilutions ofdsDNA, SYBR Green I and gluc were made with 50 mM Tris-HCl pH 7.8, 600mM NaCl, 1 mM EDTA and 20% BPER II (Pierce Biotechnology, Rockford,Ill.). Coelenterazine (Nanolight International, Pinetop, Calif.) wasdiluted into PBS, 1 mM EDTA. The final concentration of coelenterazinein the reaction was 50 uM.

Various amounts of dsDNA were preincubated with 0.78 μl of 200×concentrated SYBR Green I in a volume of 50 μl. A 1× concentration isrelative and is that defined by the manufacturer as the standard assayamount for DNA detection. Gluc (50 ng) in 5 μl was added to theprestained dsDNA. The luciferase reaction was initiated by the additionof 100 μl of coelenterazine. The reactions were performed in the wellsof a white polystyrene 96 multiwell plate (Evergreen Scientific, LosAngeles, Calif.). Light emitted by the reaction was detected with a CCDcamera (Raytest, Straubenhardt, Germany). Quantitative analysis of theimages obtained with camera was performed with the AIDA software package(Raytest) that was included with the camera. The capture of light fromthe reaction by the camera began 10 sec after the reaction wasinitiated. Light was in 1, 15 min increment (FIG. 5). Discriminationbetween light emitted by the gluc and that emitted by the excitednucleic acid stain/dsDNA duplex was made through the insertion of afilter between the light producing reaction and the aperture of the CCDcamera. The aperture setting for the camera was either 0.95 or 11. Thefilter (Clare Chemical Research, Dolores, Colo.) has been demonstratedto effectively image fluorescing nucleic acid stains that are excited bylight wavelengths in the 400 nm to 500 nm range and emit at wavelengthshigher than 500 nm when complexed with dsDNA. It does so bysignificantly filtering out wavelengths lower than 500 nm.

Results:

It was determined that under the conditions used, 50 ng of glucgenerated light levels that were within the dynamic range of detectionfor the CCD camera both with and without the filter. It was alsodetermined that the light generated by this amount of enzyme was asexpected, significantly reduced when the filter was used. This level ofenzyme was then assayed in the presence of different amounts of dsDNA(FIG. 5). FIG. 5 depicts the light generating reaction performed with 50ng of gluc and coelenterazine in the presence of SYBR Green 1 and 0 μg,0.063 μg, 0.125 μg, 0.25 μg, 0.5 μg, 1 μg, and 2 μg of linearized dsDNA(spots 1-7 respectively). Part A shows the CCD camera images for thereactions with filter. Part B shows the same data as relative intensityper spot.

In this experiment it was clearly shown that when both the luciferaseenzyme and nucleic acid stain were held constant and the amount of dsDNAwas increased, the light produced at longer wavelengths increasedproportionally. This demonstrates the ability of gluc to act as anintrinsic light source to activate nucleic acid intercalated dye.

Example 2

Luciferase activation of a DNA intercalated dye is proportional to theamount of dye present.

Objective:

The objective of this experiment was to determine if the luciferaseenzyme of Gaussia princeps (gluc) is sufficient to act as an excitationsource for a fluorophore that is staining double stranded (ds) DNA.Specifically, the experiment is intended to determine if the gluc whichemits light at a peak of 480 nm can excite a dsDNA intercalated nucleicacid stain with an excitation maximum in the range of 495 nm to 500 nmand an emission maximum of approximately 520 nm. This would be done bydetecting light from a gluc, fluorescing nucleic acid stain/dsDNAmixture which has had wavelengths below 500 nm filtered out.

Materials and Methods:

The reactions were performed with the same reagents and under the sameconditions as described in Example 1. However, in this experiment theconcentrations of dsDNA and gluc are held constant and the concentrationof SYBR Green I is varied (FIG. 6).

dsDNA (2 ug) was preincubated with SYBR Green I in a volume of 50 μl tofinal concentrations of 0×, 0.16×, 0.3×, 0.63×, 1.3×, 2.5×, 5×, and 10×.Gluc (50 ng) in 5 μl was added to the prestained dsDNA. The luciferasereaction was initiated by the addition of 100 μl coelenterazine.

The amount of light emitted over 500 nm in each reaction was determinedas described in Example 1. The same reactions were also performed in theabsence of dsDNA (−dsDNA).

Results:

It was determined that under the conditions used, 50 ng of glucgenerated light levels that were within the dynamic range of detectionfor the CCD camera both with and without the filter. It was alsodetermined that the light generated by this amount of enzyme was asexpected, significantly reduced when the filter was used. This level ofenzyme was then assayed in the presence of different amounts of SYBRGreen I (FIG. 6). FIG. 6 depicts the light generating reaction performedwith 50 ng of gluc and coelenterazine in the presence (+dsDNA) orabsence (−dsDNA) of linearized dsDNA and SYBR Green Ito finalconcentrations of 0×, 0.16×, 0.3×, 0.63×, 1.3×, 2.5×, 5×, and 10× (spots1-8 respectively). Part A shows the CCD camera images for the reactionswith filter. Part B shows the same data as relative intensity per spot.

In this experiment it was clearly shown that when both the luciferaseenzyme and dsDNA were held constant and the amount of SYBR Green I wasincreased, the light produced at longer wavelengths increasedproportionally. This demonstrates the ability of gluc to act as anintrinsic light source to activate dsDNA duplex intercalated fluorescingdye.

Example 3 Proximity Dependent Activation of a dsDNA Intercalated DyeObjective

The purpose of this experiment was to demonstrate the dependence thatproximity of the Chemiluminescent Molecule to the nucleic acidintercalated dye has on activation of the dye. In this experimentbiotinylated dsDNA target is mixed with biotinylated gluc. In thismixture there is no association of the two species of molecules with oneanother. Upon addition of increasing amounts of streptavidin the dsDNAand gluc become associated with one another with the streptavidin actingas an intermediate (FIG. 7A). This is due to the tight non-covalentbinding of the biotin moieties on the dsDNA and gluc to the fouravailable biotin-binding sites on the streptavidin. As the amount ofstreptavidin increases the greater the number of molecules of dsDNA thatare placed in close proximity to the gluc molecules also increases. Ifthe activation of the intercalated dye is dependent on the closeproximity of the gluc to the dye then the amount of fluorescence atlonger wavelengths should increase as the amount of streptavidinincreases.

Materials and Methods

The reactions were performed with the same reagents and under the sameconditions as described in Example 1. However, in this experiment thedsDNA used was made by annealing two complementary synthetic(Sigma-Genosys, The Woodlands, Tex.) oligonucleotides of DNA 85 and 95nucleotides in length. One of the oligomers (95 nucleotides) wasbiotinylated at the 5′ end. Streptavidin was from Pierce Biotechnology(Rockford, Ill.).

dsDNA (2.5 pmole of biotinylated 5′ end per reaction) was preincubatedwith 0.78 μl of 200×SYBR Green I in a volume of 50 μl. Gluc (50 ng) in 5μl was added to the prestained dsDNA. Streptavidin in various amounts in5 μl ddH2O was added to this mix. The mix was incubated 15 min withgentle shaking at room temperature. The luciferase reaction wasinitiated by the addition of 100 μl of coelenterazine. The amount oflight emitted over 500 nm in each reaction was determined as describedin Example 1 (FIG. 7B, FIG. 7C).

Results

FIG. 7 depicts the light generating reaction performed with 50 ng ofbiotinylated gluc and coelenterazine in the presence of PicoGreen dye.The DNA target for each reaction was at concentration of 2.5 pmole perbiotinylated end per reaction. Streptavidin was present at 0.013 pmole,0.026 pmole, 0.05 pmole, 0.1 pmole, 0.2 pmole, 0.42, 0.84 pmole (spots1-8 respectively). Part A shows a schematic diagram of the experimentaldesign. Part B shows the CCD camera images for the reactions withfilter. Part C shows the CCD camera images for each reaction assessed asrelative intensity per spot. In this experiment it was shown that theamount of light that can pass through the filter to the CCD camera isdirectly proportional to the amount of streptavidin that is added. Allother components, luciferase, intercalating dye, dsDNA, andcoelenterazine are the same in each reaction. The streptavidin serves tobring the Chemiluminescent Molecule (gluc), and stained nucleic acidinto a single complex in close proximity to one another. As morestreptavidin is added more of the complex is created. The increase incomplex is directly proportional the amount of longer wavelength light.

Example 4

FIG. 8 depicts a hypothetical experiment representing a furtherapplication of the method. Each data point represents the intensity of alight emitting reaction with the amount of single stranded DNA targetincreasing in each reaction going from left to right. Gluc/DNA probe,PICOGREEN® and coelenterazine are held constant. Reactions are witheither a) Probe with sequence complementary to the target DNA or b)Probe with sequence not complementary to the target DNA.

1. A method comprising: a) providing a sample suspected of containing anucleic acid of interest, wherein said nucleic acid of interest iscovalently or non-covalently attached to a chemiluminescent molecule,wherein said chemiluminescent molecule is selected from the groupconsisting of luciferase, firefly luciferase, alkaline phosphatase, andhorseradish peroxidase; b) contacting the sample with a nucleic acidmolecule complementary to the nucleic acid of interest to form a nucleicacid duplex molecule; c) contacting the sample with an intercalating dyeto generate a dye-bound nucleic acid duplex; d) activating saidchemiluminescent molecule to produce light, wherein the light excitesthe dye bound nucleic acid duplex, wherein said activating is initiatedby adding a chemiluminescent substrate to the sample, and whereby nolight source other than the chemiluminescent molecule is used; and e)detecting the light emitted by the excited dye bound nucleic acidduplex, whereby a nucleic acid of interest is detected.
 2. (canceled) 3.A method comprising: a) providing a sample suspected of containing anucleic acid of interest; b) contacting the sample with a nucleic acidmolecule complementary to the nucleic acid of interest to form a nucleicacid duplex, wherein said nucleic acid complementary to the nucleic acidof interest is covalently or non-covalently attached to achemiluminescent molecule, wherein said chemiluminescent molecule isselected from the group consisting of luciferase, firefly luciferase,alkaline phosphatase, and horseradish peroxidase; c) contacting thesample with an intercalating dye to generate a dye-bound nucleic acidduplex; d) activating said chemiluminescent molecule to produce light,wherein the light excites the dye bound nucleic acid duplex, whereinsaid activating is initiated by adding a chemiluminescent substrate tothe sample, and wherein no light source other than the chemiluminescentmolecule is used; and e) detecting the light emitted by theintercalating dye, whereby a nucleic acid of interest is detected. 4.The method according to claim 1 or 3, wherein said nucleic acid ofinterest is selected from the group consisting of DNA, RNA, andderivative thereof.
 5. The method according to claim 1 or 3, whereinsaid nucleic acid molecule complementary to the nucleic acid of interestis selected from the group consisting of DNA, RNA, and derivativethereof.
 6. The method according to claim 1 or 3, wherein saidchemiluminescent molecule is activated by a luciferin.
 7. The method ofclaim 1 or 3, wherein said chemiluminescent molecule is activated by aluciferin selected from the group consisting of firefly luciferin,coelenterazine, bacterial luciferin, dinoflagellate luciferin, vargulin,and synthetic analogs of the foregoing that are oxidized in the presenceof a luciferase in a reaction that produces bioluminescence.
 8. Themethod according to claim 1 or 3, wherein at least one of the nucleicacids of interest and the complementary nucleic acid is bound to a solidsupport.
 9. The method according to claim 1 or 3, wherein theintercalating dye is selected from the group consisting of2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]and2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium.10. The method according to claim 1 or 3, wherein detecting the light isqualitative.
 11. The method according to claim 1 or 3, wherein detectingthe light is quantitative.
 12. The method according to claim 1 or 3,wherein said chemiluminescent molecule is a luciferase.
 13. The methodaccording to claim 1 or 3, wherein said chemiluminescent molecule is aluciferase from a system selected from the group consisting of Renilla,Gaussia, Pleuromamma, Aequorea, Obelia, Porichthys, Aristostomias,Odontosyllis, Oplophorus, firefly, bacterial, Cavarnularia, Ptilosarcus,Stylatula, Acanthoptilum, Parazoanthus, Chiroteuthis, Eucleoteuthis,Onychoteuthis, Watasenia; cuttlefish, Sepiolina, Oplophorus, Sergestes,Gnathophausia; Argyropelecus, Yarella, Diaphus, and Neoscopelus systems.14. The method according to claim 1 or 3, wherein said chemiluminescentmolecule is Gaussia princeps luciferase.
 15. The method of claim 3,wherein said chemiluminescent molecule is associated with saidcomplementary nucleic acid molecule by means selected from the groupconsisting of covalent and non-covalent interactions.
 16. The method ofclaim 1, wherein said chemiluminescent molecule is associated with saidnucleic acid of interest by means selected from the group consisting ofcovalent and non-covalent interactions.
 17. A method comprising: a)providing a sample suspected of containing a nucleic acid of interest,b) contacting the sample with a nucleic acid molecule complementary tothe nucleic acid of interest to form a nucleic acid duplex, wherein thenucleic acid duplex incorporates fluorescently labeled nucleotides ornucleotide analogs that fluoresce; c) contacting the sample with achemiluminescent molecule, wherein said chemiluminescent molecule isselected from the group consisting of luciferase, firefly luciferase,alkaline phosphatase, and horseradish peroxidase; d) activating thechemiluminescent molecule to produce light, wherein the light excitesthe fluorescently labeled nucleotides or nucleotide analogs thatfluoresce, and wherein said activating is by adding a chemiluminescentsubstrate into the sample and no light source other than thechemiluminescent molecule is used; and e) detecting the light emitted bythe fluorescently labeled nucleotides or nucleotide analogs thatfluoresce, whereby said nucleic acid of interest is detected.
 18. Amethod comprising: a) providing a sample suspected of containing anucleic acid of interest, wherein said nucleic of interest is covalentlyor non-covalently attached to a Gaussia luciferase; b) contacting thesample with a nucleic acid molecule complementary to the nucleic acid ofinterest to form a nucleic acid duplex molecule; c) contacting thesample with an intercalating dye selected from the group consisting of2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]and2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinoliniumto generate a dye-bound nucleic acid duplex; d) adding coelenterazine tothe sample to activate said Gaussia luciferase to produce light thatexcites the dye-bound nucleic acid duplex; and e) detecting the lightemitted by the excited dye-bound nucleic acid duplex, whereby a nucleicacid of interest is detected.
 19. A method comprising: a) providing asample suspected of containing a nucleic acid of interest; b) contactingthe sample with a nucleic acid molecule complimentary to the nucleicacid of interest to form a nucleic acid duplex, wherein said nucleicacid complementary to the nucleic acid of interest is covalently ornon-covalently attached to a Gaussia luciferase; c) contacting thesample with an intercalating dye selected from the group consisting of2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]and2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinoliniumto generate a dye-bound nucleic acid duplex; d) adding coelenterazine tothe sample to activate said Gaussia luciferase to produce light thatexcites the dye-bound nucleic acid duplex; and e) detecting the lightemitted by the excited dye-bound nucleic acid duplex, whereby a nucleicacid of interest is detected.